Transgenic corn event MON87403 and methods for detection thereof

ABSTRACT

The present disclosure provides a transgenic corn comprising event MON87403 that exhibits increased grain yield. The disclosure also provides cells, plant parts, seeds, plants, commodity products related to the event, and DNA molecules that are unique to the event and were created by the insertion of transgenic DNA into the genome of a corn plant. The disclosure further provides methods for detecting the presence of said corn event nucleotide sequences in a sample, probes and primers for use in detecting nucleotide sequences that are diagnostic for the presence of said corn event.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/516,008, filed Jul. 18, 2019, which is a divisional of U.S.application Ser. No. 16/160,841, filed Oct. 15, 2018, now U.S. Pat. No.10,407,741, which is a divisional of U.S. application Ser. No.15/289,635, filed Oct. 10, 2016, now U.S. patent Ser. No. 10/233,507,which is a divisional of U.S. application Ser. No. 14/507,734, filedOct. 6, 2014, now U.S. Pat. No. 9,469,880, which claims the benefit ofU.S. Provisional Application No. 61/888,978, filed Oct. 9, 2013, each ofwhich is herein incorporated by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“MONS342USP1_ST25.txt,” which is 27 kilobytes as measured in MicrosoftWindows operating system and was created on Oct. 4, 2013, is filedelectronically herewith and incorporated herein by reference.

FIELD

The present disclosure relates to transgenic corn event MON87403 andplants comprising the event that exhibit increased yield. The disclosurealso provides cells, plant parts, seeds, plants, commodity productsrelated to the event, and DNA molecules that are unique to the event andwere created by the insertion of transgenic DNA into the genome of acorn plant. The disclosure further provides methods for detecting thepresence of said corn event nucleotide sequences in a sample, probes andprimers for use in detecting nucleotide sequences that are diagnosticfor the presence of said corn event.

BACKGROUND

Corn is an important crop and is a primary food source in many areas ofthe world. The methods of biotechnology have been applied to corn forimprovement of agronomic traits and the quality of the product. One suchagronomic trait is increased yield.

Increased yield may be achieved in transgenic plants by the expressionof a transgene capable of providing such increased yield. The expressionof transgenes in plants may be influenced by many factors, such as theregulatory elements used in the transgene cassette, the chromosomallocation of the transgene insert, the proximity of any endogenousregulatory elements close to the transgene insertion site, andenvironmental factors such as light and temperature. For example, theremay be a wide variation in the overall level of transgene expression orin the spatial or temporal pattern of transgene expression betweensimilarly-produced events. For this reason, the performance of a singlegiven transformation event can vary. The identification oftransformation events conferring beneficial characteristics cantherefore represent a significant undertaking.

SUMMARY

In one aspect, the invention provides a recombinant DNA moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1-8, SEQ ID NO:10, and full complements thereof. In oneembodiment, the recombinant DNA molecule is formed by the junction of aninserted heterologous nucleic acid molecule and genomic DNA of a cornplant, plant cell, or seed. In another embodiment, the recombinant DNAmolecule is from a transgenic corn plant comprising event MON87403, arepresentative sample of seed comprising said event having beendeposited as ATCC Accession No. PTA-13584. In another embodiment, therecombinant DNA molecule is an amplicon diagnostic for the presence ofDNA from transgenic corn event MON87403. In still another embodiment,the recombinant DNA molecule is in a corn plant, plant cell, seed,progeny plant, plant part, or commodity product. In another embodiment,the invention provides a transgenic corn plant, seed, cell, or plantpart thereof comprising the recombinant DNA molecule. In otherembodiments, the transgenic corn plant, seed, cell, or plant partthereof comprising the recombinant DNA molecule has increased yield,and/or the genome of such corn plant, seed, cell, or plant part thereofproduces an amplicon comprising a DNA molecule selected from the groupconsisting of SEQ ID NO:1-8, and consecutive nucleotides of SEQ ID NO:10when tested in a DNA amplification method. In another embodiment, theinvention provides a nonliving plant material and/or a microorganismcomprising the recombinant DNA molecule. In another embodiment, themicroorganism comprising the recombinant DNA molecule is a plant cell.

In another aspect, the invention provides a DNA probe comprising anucleotide sequence of sufficient length of contiguous nucleotides ofSEQ ID NO:10, or a full complement thereof, to function as a DNA probethat hybridizes under stringent hybridization conditions with a DNAmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1-8, and SEQ ID NO:10 and does not hybridizeunder the stringent hybridization conditions with a DNA molecule notcomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1-8, and SEQ ID NO:10.

In another aspect, the invention provides a pair of DNA moleculescomprising a first DNA molecule and a second DNA molecule different fromthe first DNA molecule, wherein said first and second DNA molecules eachcomprise a nucleotide sequence of sufficient length of contiguousnucleotides of SEQ ID NO:10, or a full complement thereof, to functionas DNA primers when used together in an amplification reaction with DNAfrom event MON87403 to produce an amplicon diagnostic for transgeniccorn event MON87403 DNA in a sample.

In still another aspect, the invention provides a method of detectingthe presence of a DNA molecule from a transgenic corn plant comprisingevent MON87403 in a sample, said method comprising: (a) contacting saidsample with the DNA probe of claim 6; (b) subjecting said sample andsaid DNA probe to stringent hybridization conditions; and (c) detectinghybridization of said DNA probe to a DNA molecule in said sample,wherein the hybridization of said DNA probe to said DNA moleculeindicates the presence of a DNA molecule from a transgenic corn plantcomprising event MON87403 in said sample.

Another aspect of the invention provides a method of detecting thepresence of a DNA molecule from a transgenic corn plant comprising eventMON87403 in a sample, said method comprising: (a) contacting said samplewith the pair of DNA molecules of claim 7; (b) performing anamplification reaction sufficient to produce a DNA amplicon comprising asequence selected from the group consisting of SEQ ID NO:1-8 andconsecutive nucleotides of SEQ ID NO:10; and (c) detecting the presenceof said DNA amplicon in said reaction, wherein the presence of said DNAamplicon in said reaction indicates the presence of a DNA molecule froma transgenic corn plant comprising event MON87403 in said sample.

In another aspect, the invention provides a DNA detection kitcomprising: (a) a pair of DNA molecules comprising a first DNA moleculeand a second DNA molecule different from the first DNA molecule, whereinsaid first and second DNA molecules each comprise a nucleotide sequenceof sufficient length of contiguous nucleotides of SEQ ID NO:10, or afull complement thereof, to function as DNA primers when used togetherin an amplification reaction with DNA from event MON87403 to produce anamplicon diagnostic for transgenic corn event MON87403 DNA; and (b) aDNA probe comprising a nucleotide sequence of sufficient length ofcontiguous nucleotides of SEQ ID NO:10, or a full complement thereof, tofunction as a DNA probe that hybridizes under stringent hybridizationconditions with a DNA molecule comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:1-8 and SEQ ID NO:10 and does nothybridize under the stringent hybridization conditions with a DNAmolecule not comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1-8 and SEQ ID NO:10.

Another aspect of the present invention provides a corn plant or seed,comprising event MON87403, a representative sample of seed comprisingsaid event having been deposited under ATCC Accession No. PTA-13584. Inone embodiment, the corn plant or seed is a hybrid having at least oneparent comprising event MON87403.

In still another aspect, the invention provides a commodity productproduced from a transgenic corn plant comprising event MON87403 andcomprising the recombinant DNA molecule of claim 1, wherein detection ofsaid nucleotide sequence in a sample derived from said commodity productis determinative that said commodity product was produced from saidtransgenic corn plant comprising event MON87403. In one embodiment, thecommodity product is selected from the group consisting of whole orprocessed seeds, animal feed, oil, meal, flour, flakes, bran, biomass,and fuel products. Other embodiments of the invention provide a methodof producing the commodity product, comprising: (a) obtaining a cornplant or part thereof comprising transgenic corn event MON87403; and (b)producing a corn commodity product from the corn plant or part thereof.

Another aspect of the invention provides a method of increasing yield ina crop comprising: (a) planting a crop plant or seed comprising eventMON87403; and (b) growing said crop plant or seed. In one embodiment,the crop plant or seed is a corn plant or corn seed.

In another aspect, the invention provides a method of producing a cornplant with increased yield comprising: (a) sexually crossing atransgenic corn plant comprising event MON87403 comprising a nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1-8, consecutive nucleotides of SEQ ID NO:10,and full complements thereof, with a second maize plant, therebyproducing seed; (b) collecting said seed produced from said cross; (c)growing said seed to produce a plurality of progeny plants; and (d)selecting a progeny plant that has increased yield.

In another aspect, the invention provides a method of producing a cornplant with increased yield comprising: (a) selfing a transgenic cornplant comprising event MON87403 comprising a nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting ofSEQ ID NO:1-8, and consecutive nucleotides of SEQ ID NO:10, therebyproducing seed; (b) collecting said seed produced from said selfing; (c)growing said seed to produce a plurality of progeny plants; and (d)selecting a progeny plant that has increased yield.

Another aspect of the invention provides a method of producing hybridcorn seed comprising: (a) planting transgenic corn seed comprising eventMON87403 in an area; (b) growing a corn plant from said seed; (c)fertilizing said corn plant with pollen from a second parent corn plant;and (d) harvesting seed from said corn plant, wherein said seed ishybrid corn seed produced by the cross of a transgenic corn plantcomprising event MON87403 with a second parent plant. In one embodiment,the method further comprising planting a second parent corn plant seedin said area and growing a corn plant from said second parent cornplant. In another embodiment, the said second parent corn plant hasincreased yield.

In another aspect, the invention provides a method of determining thezygosity of a corn plant genome comprising corn event MON87403 DNA in asample comprising: (a) contacting the sample with a first pair of DNAmolecules and a second distinct pair of DNA molecules that: (i) whenused together in a nucleic acid amplification reaction with corn eventMON87403 DNA, produces a first amplicon that is diagnostic for cornevent MON87403; and (ii) when used together in a nucleic acidamplification reaction with corn genomic DNA other than MON87403 DNA,produces a second amplicon that is diagnostic for corn wild type genomicDNA other than event MON87403 DNA; (b) performing a nucleic acidamplification reaction; and (c) detecting said first amplicon and saidsecond amplicon; wherein the presence of said first and second ampliconsis diagnostic of a heterozygous genome in said sample, and wherein thepresence of only said first amplicon is diagnostic of a genomehomozygous for corn event MON87403 in said sample. In an embodiment, thefirst set of DNA molecules comprises SEQ ID NO:11 and SEQ ID NO:12, andthe second set of DNA molecules comprises SEQ ID NO:14 and SEQ ID NO:15.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Shows a diagrammatical representation of the transgenic insert inthe genome of a corn comprising event MON87403; [A] corresponds to therelative positions of SEQ ID NOs:1, 3, and 5, all of which form thejunction between the 5′ portion of the transgenic insert and the 3′portion of the flanking genomic DNA; [B] corresponds to the relativepositions of SEQ ID NOs:2, 4, and 6, all of which form the junctionbetween the 3′ portion of the transgenic insert and the 5′ portion ofthe flanking genomic DNA; [C] corresponds to the relative position ofSEQ ID NO:7, which contains the corn genomic flanking region and aportion of the arbitrarily designated 5′ end of the transgenic DNAinsert; [D] corresponds to the relative position of SEQ ID NO:8, whichcontains the corn genome flanking region and a portion of thearbitrarily designated 3′ end of the transgenic DNA insert; [E]represents SEQ ID NO:9, which is the sequence of the transgenic DNAinsert including the ATHB17 expression cassette integrated into thegenome of a corn plant comprising event MON87403; [F] represents SEQ IDNO:10, which is the contiguous sequence comprising the 5′ flankinggenomic sequence, the transgenic insert and the 3′ flanking genomicsequence, comprising, as represented in the figure from left to right,SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:8, in which SEQ ID NOs:1, 3, and5, and SEQ ID NOs:2, 4, and 6 are incorporated as set forth above, asthese sequences are present in the genome of a plant comprising eventMON87403.

FIG. 2—Shows comparative data of 14 individual events that were advancedto field testing in 2009. Data shown are from the 2009 multi-testertrial taken from 11 locations, with 22 total replicates. Asterisksindicate an event ranked in the top 5.

FIG. 3—Shows the plasmid map of transformation vector pMON97046.

FIG. 4—Shows the increased yield performance of plants comprising theMON87403 event in transformation germplasm-based hybrids across years.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a 20 nucleotide sequence representing the 5′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette (positions 1336 through 1355 of SEQ ID NO:10).

SEQ ID NO:2 is a 20 nucleotide sequence representing the 3′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette (positions 4468 through 4487 of SEQ ID NO:10).

SEQ ID NO:3 is a 60 nucleotide sequence representing the 5′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette (positions 1316 through 1375 of SEQ ID NO:10).

SEQ ID NO:4 is a 60 nucleotide sequence representing the 3′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette (positions 4448 through 4507 of SEQ ID NO:10).

SEQ ID NO:5 is a 100 nucleotide sequence representing the 5′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette (positions 1296 through 1395 of SEQ ID NO:10).

SEQ ID NO:6 is a 100 nucleotide sequence representing the 3′ junctionregion of a maize genomic DNA and an integrated transgenic expressioncassette (positions 4428 through 4527 of SEQ ID NO:10).

SEQ ID NO:7 is a 3000 nucleotide 5′ sequence flanking the inserted DNAof MON87403 up to and including a region of transgene DNA insertion(positions 1 through 3000 of SEQ ID NO:10).

SEQ ID NO:8 is a 2624 nucleotide 3′ sequence flanking the inserted DNAof MON87403 up to and including a region of transgene DNA insertion(positions 3121 through 5744 of SEQ ID NO:10).

SEQ ID NO:9 is the sequence fully integrated into the maize genomic DNAand containing the expression cassette DNA (positions 1346 through 4477of SEQ ID NO:10).

SEQ ID NO:10 is the nucleotide sequence representing the contig of the5′ sequence flanking the inserted DNA of MON87403 (SEQ ID NO:7), thesequence fully integrated into the corn genomic DNA and containing theexpression cassette (SEQ ID NO:9), and the 3′ sequence flanking theinserted DNA of MON87403 (SEQ ID NO:8) and includes SEQ ID NOs:1-6.

SEQ ID NO:11 is a transgene-specific assay primer SQ23846 used toidentify event MON87403. A PCR amplicon produced from a TAQMAN® (PEApplied Biosystems, Foster City, Calif.) assay using the combination ofprimers SEQ ID NO:11 and SEQ ID NO:12 is a positive result for thepresence of the event MON87403.

SEQ ID NO:12 is a transgene-specific assay primer SQ4603 used toidentify event MON87403.

SEQ ID NO:13 is a transgene-specific assay 6-FAM-labeled probe PB10644used to identify MON87403. This probe is a 6FAM™-labeled syntheticoligonucleotide. Release of a fluorescent signal in an amplificationreaction using primers SEQ ID NO:11-12 in combination with the6FAM™-labeled probe is diagnostic of event MON87403 in a TAQMAN® assay.

SEQ ID NO:14 is a transgene-specific assay internal control primerSQ25061.

SEQ ID NO:15 is a transgene-specific assay internal control primerSQ25062.

SEQ ID NO:16 is a transgene-specific assay internal control VIC™-labeledPB10866.

SEQ ID NO:17 is a transgene-specific forward primer AS349 for PCR usedto identify MON87403.

SEQ ID NO:18 is a transgene-specific reverse primer AS350 for PCR usedto identify MON87403.

SEQ ID NO:19 is primer SQ6164, used to detect the 5′ (left) junctionregion of event MON87403.

SEQ ID NO:20 is primer SQ13205, used in PCR to detect the 5′ (left)junction region of event MON87403.

SEQ ID NO:21 is primer SQ6165, used to detect the 5′ (left) junctionregion of event MON87403.

SEQ ID NO:22 is primer SQ22458, used to detect the 5′ (left) junctionregion of event MON87403.

SEQ ID NO:23 is primer SQ22459, used to detect the 5′ (left) junctionregion of event MON87403.

SEQ ID NO:24 is primer SQ21173, used to detect the 3′ (right) junctionregion of event MON87403.

SEQ ID NO:25 is primer SQ22464, used to detect the 3′ (right) junctionregion of event MON87403.

SEQ ID NO:26 is primer SQ22460, used to detect the 3′ (right) junctionregion of event MON87403.

SEQ ID NO:27 is primer SQ22465, used to detect the 3′ (right) junctionregion of event MON87403.

SEQ ID NO:28 is primer SQ22461, used to detect the 3′ (right) junctionregion of event MON87403.

SEQ ID NO:29 is primer SQ22471, used to detect the 3′ (right) junctionregion of event MON87403.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent disclosure and to guide those of ordinary skill in the art inthe practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art. Definitions of common terms inmolecular biology may also be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

The present disclosure provides transgenic corn event MON87403 (alsoreferred to herein as MON87403). The term “event” as used herein refersto DNA molecules produced as a result of inserting transgenic DNA into aplant's genome at a particular location on a chromosome. Event MON87403refers to the DNA molecules produced as a result of the insertion oftransgenic DNA having a sequence provided herein as SEQ ID NO:9 into aparticular chromosomal location in the Zea mays genome. Plants, seeds,progeny, cells, and plant parts thereof comprising event MON87403 arealso provided in the present disclosure. Plants comprising MON87403exhibit increased grain yield.

As used herein, the term “corn” or “maize” means Zea mays and includesall plant varieties that can be bred with maize, including wild maizespecies as well as those plants belonging to Zea that permit breedingbetween species.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that comprises atransgene of interest, regeneration of a population of independentlytransformed transgenic plants resulting from the insertion of thetransgene into the genome of the plants, and selection of a particularplant with desirable molecular characteristics, such as insertion of asingle copy of the transgene into a particular genome location,integrity of the transgenic DNA, and an enhanced trait such as increasedgrain yield. A plant comprising the event can refer to the originaltransformant that includes the transgene inserted into the particularlocation in the plant's genome. A plant comprising the event can alsorefer to progeny of the original transformant that retain the transgeneat the same particular location in the plant's genome. Such progeny maybe produced by selfing, or by a sexual outcross with a different plantcomprising the same event, or its progeny, and another plant. Suchanother plant may be a transgenic plant comprising the same or adifferent transgene; or may be a non-transgenic plant, such as one froma different variety. The resulting progeny may be homozygous orheterozygous for event MON87403 DNA (inserted DNA and flanking DNA).Even after repeated back-crossing to a recurrent parent, the event DNAfrom the transformed parent is present in the progeny of the cross atthe same genomic location.

A DNA molecule comprising event MON87403 refers to a DNA moleculecomprising at least a portion of the inserted transgenic DNA (providedas SEQ ID NO:9) and at least a portion of the flanking genomic DNAimmediately adjacent to the inserted DNA. As such, a DNA moleculecomprising event MON87403 has a nucleotide sequence representing atleast a portion of the transgenic DNA insert and at least a portion ofthe particular region of the genome of the plant into which thetransgenic DNA was inserted. The arrangement of the inserted DNA inevent MON87403 in relation to the surrounding plant genome is specificand unique to event MON87403 and as such the nucleotide sequence of sucha DNA molecule is diagnostic and identifying for event MON87403.Examples of the sequence of such a DNA molecule are provided herein asSEQ ID NOs:1-8 and SEQ ID NO:10. Such a DNA molecule is also an integralpart of the chromosome of a plant that comprises event MON87403 and maybe passed on to progeny of the plant.

As used herein, a “recombinant DNA molecule” is a DNA moleculecomprising a combination of DNA molecules that would not naturally occurtogether and is the result of human intervention, e.g., a DNA moleculethat is comprised of a combination of at least two DNA moleculesheterologous to each other, and/or a DNA molecule that is artificiallysynthesized and comprises a polynucleotide sequence that deviates fromthe polynucleotide sequence that would normally exist in nature, and/ora DNA molecule that comprises a transgene incorporated into a hostcell's genomic DNA and the associated flanking DNA of the host cell'sgenome. An example of a recombinant DNA molecule is a DNA moleculedescribed herein resulting from the insertion of the transgene into theZea mays genome, which may ultimately result in the expression of arecombinant RNA and/or protein molecule in that organism. The nucleotidesequence or any fragment derived therefrom would also be considered arecombinant DNA molecule if the DNA molecule can be extracted fromcells, or tissues, or homogenate from a plant or seed or plant tissue;or can be produced as an amplicon from extracted DNA or RNA from cells,or tissues, or homogenate from a plant or seed or plant tissue, any ofwhich is derived from such materials derived from a plant comprisingevent MON87403. For that matter, the junction sequences as set forth atSEQ ID NOs:1-6, and nucleotide sequences derived from event MON87403that also contain these junction sequences are defined herein to berecombinant DNA, whether these sequences are present within the genomeof the cells comprising event MON87403 or present in detectable amountsin tissues, progeny, biological samples or commodity products derivedfrom plants comprising event MON87403. As used herein, the term“transgene” refers to a polynucleotide molecule incorporated into a hostcell's genome. Such transgene may be heterologous to the host cell. Theterm “transgenic plant” refers to a plant comprising such a transgene. A“transgenic plant” includes a plant, plant part, a plant cell or seedwhose genome has been altered by the stable integration of recombinantDNA. A transgenic plant includes a plant regenerated from anoriginally-transformed plant cell and progeny transgenic plants fromlater generations or crosses of a transformed plant. As a result of suchgenomic alteration, the transgenic plant is distinctly different fromthe related wild type plant. An example of a transgenic plant is a plantdescribed herein as comprising event MON87403.

As used herein, the term “heterologous” refers to a sequence that is notnormally present in a given host genome in the genetic context in whichthe sequence is currently found. In this respect, the sequence may benative to the host genome, but be rearranged with respect to othergenetic sequences within the host sequence.

The present disclosure provides DNA molecules and their correspondingnucleotide sequences. As used herein, the terms “DNA sequence,”“nucleotide sequence,” and “polynucleotide sequence” refer to thesequence of nucleotides of a DNA molecule, usually presented from the 5′(upstream or left) end to the 3′ (downstream or right) end. Thenomenclature used herein is that required by Title 37 of the UnitedStates Code of Federal Regulations § 1.822 and set forth in the tablesin WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. The presentdisclosure is disclosed with reference to only one strand of the twonucleotide sequence strands that are provided in transgenic eventMON87403. Therefore, by implication and derivation, the complementarysequences, also referred to in the art as the complete complement or thereverse complementary sequences, are within the scope of the presentdisclosure and are therefore also intended to be within the scope of thesubject matter claimed.

The nucleotide sequence corresponding to the complete nucleotidesequence of the inserted transgenic DNA and substantial segments of theZea mays genomic DNA flanking either end of the inserted transgenic DNAis provided herein as SEQ ID NO:10. A subsection of this is the insertedtransgenic DNA provided as SEQ ID NO:9. The nucleotide sequence of thegenomic DNA flanking the 5′ end of the inserted transgenic DNA and aportion of the 5′ end of the inserted DNA is provided herein as SEQ IDNO:7. The nucleotide sequence of the genomic DNA flanking the 3′ end ofthe inserted transgenic DNA and a portion of the 3′ end of the insertedDNA is provided herein as SEQ ID NO:8. The region spanning the locationwhere the transgenic DNA connects to and is linked to the genomic DNA isreferred to herein as the junction. A “junction sequence” or “junctionregion” refers to a DNA sequence and/or corresponding DNA molecule thatspans the inserted transgenic DNA and the adjacent flanking genomic DNA.Examples of a junction sequence of event MON87403 are provided herein asSEQ ID NOs:1-6. The identification of one of these junction sequences ina nucleotide molecule derived from a corn plant or seed is conclusivethat the DNA was obtained from event MON87403 and is diagnostic for thepresence of DNA from event MON87403. SEQ ID NO:1 is a 20 bp nucleotidesequence spanning the junction between the genomic DNA and the 5′ end ofthe inserted DNA. SEQ ID NO:3 is a 60 bp nucleotide sequence spanningthe junction between the genomic DNA and the 5′ end of the inserted DNA.SEQ ID NO:5 is a 100 bp nucleotide sequence spanning the junctionbetween the genomic DNA and the 5′ end of the inserted DNA. SEQ ID NO:2is a 20 bp nucleotide sequence spanning the junction between the genomicDNA and the 3′ end of the inserted DNA. SEQ ID NO:4 is a 60 bpnucleotide sequence spanning the junction between the genomic DNA andthe 3′ end of the inserted DNA. SEQ ID NO:6 is a 100 bp nucleotidesequence spanning the junction between the genomic DNA and the 3′ end ofthe inserted DNA. Any segment of DNA derived from transgenic eventMON87403 that includes at least 19 consecutive nucleotides of SEQ IDNO:1, or 31, 32, 33, 34, 35, 40, 45, 50, 55, or all consecutivenucleotides of SEQ ID NO:3, or 51, 52, 53, 54, 55, 60, 65, 70, 75, 80,85, 90, 95, or all consecutive nucleotides of SEQ ID NO:5 is within thescope of the present disclosure. Any segment of DNA derived fromtransgenic event MON87403 that includes at least 18 consecutivenucleotides of SEQ ID NO:2, or 31, 32, 33, 34, 35, 40, 45, 50, 55, orall consecutive nucleotides of SEQ ID NO:4, or 51, 52, 53, 54, 55, 60,65, 70, 75, 80, 85, 90, 95, or all consecutive nucleotides of SEQ IDNO:6 is within the scope of the present disclosure. In addition, anypolynucleotide molecule comprising a sequence complementary to any ofthe sequences described within this paragraph is within the scope of thepresent disclosure. FIG. 1 is an illustration of the transgenic DNAinsert in the genome of a corn plant comprising event MON87403, and therelative positions of SEQ ID NOs: 1-10 arranged 5′ to 3′. The presentdisclosure also provides a nucleic acid molecule comprising apolynucleotide having a sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the full-length of SEQ IDNO:10.

The present disclosure further provides exemplary DNA molecules that canbe used either as primers or probes for diagnosing the presence of DNAderived from event MON87403 in a sample. Such primers or probes arespecific for a target nucleic acid sequence and as such are useful forthe identification of event MON87403 nucleic acid sequence by themethods of the disclosure described herein.

A “probe” is an isolated nucleic acid to which is attached a detectablelabel or reporter molecule, e.g., a radioactive isotope, ligand,chemiluminescent agent, or enzyme. Such a probe is complementary to astrand of a target nucleic acid. In the case of the present disclosure,such a probe is complementary to a strand of genomic DNA from a corncomprising event MON87403, whether from a corn plant or from a samplethat comprises DNA from the event. Probes according to the presentdisclosure include not only deoxyribonucleic or ribonucleic acids butalso polyamides and other probe materials that bind specifically to atarget DNA sequence. The detection of such binding can be used todiagnose/determine/confirm the presence of that target DNA sequence in aparticular sample.

A “primer” is typically an isolated polynucleotide that is designed foruse in specific annealing or hybridization methods to hybridize to acomplementary target DNA strand to form a hybrid between the primer andthe target DNA strand, and then extended along the target DNA strand bya polymerase, e.g., a DNA polymerase. A pair of primers may be used withtemplate DNA, such as a sample of Zea mays genomic DNA, in a thermal orisothermal amplification, such as polymerase chain reaction (PCR), orother nucleic acid amplification methods, to produce an amplicon, wherethe amplicon produced from such reaction would have a DNA sequencecorresponding to sequence of the template DNA located between the twosites where the primers hybridized to the template. As used herein, an“amplicon” is a piece or fragment of DNA that has been synthesized usingamplification techniques, i.e. the product of an amplification reaction.In one embodiment of the disclosure, an amplicon diagnostic for eventMON87403 comprises a sequence not naturally found in the Zea maysgenome. Primer pairs, as used in the present disclosure, are intended torefer to use of two primers binding opposite strands of a doublestranded nucleotide segment for the purpose of amplifying linearly thepolynucleotide segment between the positions targeted for binding by theindividual members of the primer pair, typically in a thermal orisothermal amplification reaction or other nucleic acid amplificationmethods. In embodiments, exemplary DNA molecules useful as primers areprovided as SEQ ID NOs:11-12, 14-15, 17-18, and 19-29. For example,exemplary event-specific primers for PCR to identify event MON87403 areprovided as SEQ ID NOs:17-18. Exemplary primers that may be used foranalysis of the 5′ (left) junction region are provided as SEQ IDNOs:19-23, and exemplary primers that may be used for analysis of the 3′(right) junction region are provided as SEQ ID NOs:24-29. Exemplaryprimers that may be used for zygosity testing for event MON87403 areprovided as SEQ ID NOs:11-12 and SEQ ID NOs: 14-15. The use of the term“amplicon” specifically excludes primer-dimers that may be formed in theDNA amplification reaction.

Probes and primers according to the present disclosure may have completesequence identity to the target sequence, although primers and probesdiffering from the target sequence that retain the ability to hybridizepreferentially to target sequences may be designed by conventionalmethods. In order for a nucleic acid molecule to serve as a primer orprobe it needs only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed. Any nucleic acid hybridization oramplification method can be used to identify the presence of transgenicDNA from event MON87403 in a sample. Probes and primers are generally atleast about 11 nucleotides, at least about 18 nucleotides, at leastabout 24 nucleotides, and at least about 30 nucleotides or more inlength. Such probes and primers hybridize specifically to a targetsequence under high stringency hybridization conditions.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates); andInnis et al., PCR Protocols: A Guide to Methods and Applications,Academic Press: San Diego, 1990. PCR-primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose such as Primer (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.).

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm the disclosed sequences by knownmethods, e.g., by re-cloning and sequencing such sequences.

The nucleic acid probes and primers of the present disclosure hybridizeunder stringent conditions to a target DNA sequence. Any nucleic acidhybridization or amplification method can be used to identify thepresence of DNA from a transgenic event in a sample. Nucleic acidmolecules or fragments thereof are capable of specifically hybridizingto other nucleic acid molecules under certain circumstances. As usedherein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least “low-stringency”conditions. Similarly, the molecules are said to be “complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under “high-stringency”conditions. Stringency conditions are described by Sambrook et al.,1989, and by Haymes et al., In: Nucleic Acid Hybridization, A PracticalApproach, IRL Press, Washington, D.C. (1985). Departures from completecomplementarity are therefore permissible, as long as such departures donot completely preclude the capacity of the molecules to form adouble-stranded structure. In order for a nucleic acid molecule to serveas a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions that promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In oneembodiment, a nucleic acid of the present disclosure will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NOs:1-6, or complements or fragments thereof under high stringencyconditions. The hybridization of the probe to the target DNA moleculecan be detected by any number of methods known to those skilled in theart. These can include, but are not limited to, fluorescent tags,radioactive tags, antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA amplification reaction. Examples of DNA amplification methodsinclude PCR, Recombinase Polymerase Amplification (RPA) (see for exampleU.S. Pat. No. 7,485,428), Strand Displacement Amplification (SDA) (seefor example, U.S. Pat. Nos. 5,455,166 and 5,470,723),Transcription-Mediated Amplification (TMA) (see for example, Guatelli etal., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990), Rolling CircleAmplification (RCA) (see for example, Fire and Xu, Proc. Natl. Acad Sci.USA 92:4641-4645, 1995; Lui, et al., J. Am. Chem. Soc. 118:1587-1594,1996; Lizardi, et al., Nature Genetics 19:225-232, 1998; U.S. Pat. Nos.5,714,320 and 6,235,502), Helicase Dependent Amplification (HDA) (seefor example Vincent et al., EMBO Reports 5(8): 795-800, 2004; U.S. Pat.No. 7,282,328), and Multiple Displacement Amplification (MDA) (see forexample Dean et al., Proc. Natl. Acad Sci. USA 99:5261-5266, 2002).

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, the term “isolated” refers to at least partiallyseparating a molecule from other molecules normally associated with itin its native or natural state. In one embodiment, the term “isolated”refers to a DNA molecule that is at least partially separated from thenucleic acids that normally flank the DNA molecule in its native ornatural state. DNA molecules fused to regulatory or coding sequenceswith which they are not normally associated, for example as the resultof recombinant techniques, are considered isolated herein. Thus, anytransgenic, recombinant, chimeric or artificial nucleotide sequencewould be considered to be an isolated nucleotide sequence since theseare not naturally occurring sequences. A transgenic, recombinant,chimeric or artificial nucleotide sequence would be considered to be anisolated nucleotide sequence whether it is present within the plasmid,vector or construct used to transform plant cells, within the genome ofthe plant, or is present in detectable amounts in tissues, progeny,biological samples or commodity products derived from the plant. Thenucleotide sequence or any fragment derived therefrom would therefore beconsidered to be isolated or isolatable if the DNA molecule can beextracted from cells, or tissues, or homogenate from a plant or seed orplant organ; or can be produced as an amplicon from extracted DNA or RNAfrom cells, or tissues, or homogenate from a plant or seed or plantorgan, any of which is derived from such materials derived from thetransgenically altered plant.

Any number of methods well known to those skilled in the art can be usedto isolate and manipulate a DNA molecule, or fragment thereof, disclosedin the present disclosure. For example, PCR (polymerase chain reaction)technology can be used to amplify a particular starting DNA moleculeand/or to produce variants of the original molecule. DNA molecules, orfragments thereof, can also be obtained by other techniques such as bydirectly synthesizing the fragment by chemical means, as is commonlypracticed by using an automated oligonucleotide synthesizer.

It would be advantageous to be able to detect the presence oftransgene/genomic DNA of a particular plant in order to determinewhether progeny of a self-pollination or sexual cross contain thetransgene/genomic DNA of interest. In addition, a method for detecting aparticular transgenic plant is helpful when complying with regulationsrequiring the pre-market approval and labeling of foods derived from thetransgenic crop plants.

The presence of a transgene may be detected by any well known nucleicacid detection method such as the polymerase chain reaction (PCR) or DNAhybridization using nucleic acid probes. These detection methodsgenerally focus on frequently used genetic elements, such as promoters,terminators, marker genes, etc. As a result, such methods may not beuseful for discriminating between different transformation events,particularly those produced using the same DNA construct unless thesequence of chromosomal DNA adjacent to the inserted DNA (“flankingDNA”) is known. An event-specific PCR assay is discussed, for example,by Taverniers et al. (J. Agric. Food Chem., 53:3041-3052, 2005) in whichan event-specific tracing system for transgenic maize lines Bt11, Bt176,and GA21 and for canola event GT73 was demonstrated. In this study,event-specific primers and probes were designed based upon the sequencesof the genome/transgene junctions for each event. Transgenic plant eventspecific DNA detection methods have also been described in U.S. Pat.Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974; 6,689,880; 6,900,014;and 6,818,807.

The DNA molecules and corresponding nucleotide sequences provided hereinare therefore useful for, among other things, identifying eventMON87403, selecting plant varieties or hybrids comprising eventMON87403, detecting the presence of DNA derived from event MON87403 in asample, and monitoring samples for the presence and/or absence of eventMON87403 or plants and plant parts comprising event MON87403.

The present disclosure provides plants, progeny, seeds, plant cells,plant parts (such as pollen, ovule, flower, root or stem tissue, fibers,and leaves), and commodity products. These plants, progeny, seeds, plantcells, plant parts, and commodity products contain a detectable amountof a polynucleotide of the present disclosure, such as a polynucleotidecomprising at least one of the sequences provided as at least 19consecutive nucleotides of SEQ ID NO:1, at least 18 consecutivenucleotides of SEQ ID NO:2, at least 31 consecutive nucleotides of SEQID NO:3, at least 31 consecutive nucleotides of SEQ ID NO:4, at least 51consecutive nucleotides of SEQ ID NO:5, or at least 51 consecutivenucleotides of SEQ ID NO:6. Plants, progeny, seeds, plant cells, plantparts and commodity products of the present disclosure may also containone or more additional transgenes. Such transgene may be any nucleotidesequence encoding a protein or RNA molecule conferring a desirable traitincluding but not limited to increased insect resistance, increasedwater use efficiency or drought tolerance, increased yield performance,increased yield potential, increased nitrogen use efficiency or increasetolerance to nitrogen stress such as high or low nitrogen supply,increased seed quality, increased disease resistance, improvednutritional quality, and/or increased herbicide tolerance, such asglyphosate or dicamba tolerance, in which the desirable trait ismeasured with respect to a comparable plant lacking such additionaltransgene.

The present disclosure provides plants, progeny, seeds, plant cells, andplant part such as pollen, ovule kernel, flower, root or stem tissue,and leaf derived from a transgenic plant comprising event MON87403. Arepresentative sample of seed comprising event MON87403 has beendeposited according to the Budapest Treaty for the purpose of enablingthe present disclosure. The repository selected for receiving thedeposit is the American Type Culture Collection (ATCC) having an addressat 10801 University Boulevard, Manassas, Va. USA, Zip Code 20110. TheATCC repository has assigned the accession No. PTA-13584 to eventMON87403-containing seed.

The present disclosure provides a microorganism comprising a DNAmolecule having a nucleotide sequence selected from the group consistingof at least 19 consecutive nucleotides of SEQ ID NO:1, at least 18consecutive nucleotides of SEQ ID NO:2, at least 31 consecutivenucleotides of SEQ ID NO:3, at least 31 consecutive nucleotides of SEQID NO:4, at least 51 consecutive nucleotides of SEQ ID NO: 5, or atleast 51 consecutive nucleotides of SEQ ID NO:6 present in its genome.An example of such a microorganism is a transgenic plant cell.Microorganisms, such as a plant cell of the present disclosure, areuseful in many industrial applications, including but not limited to:(i) use as research tool for scientific inquiry or industrial research;(ii) use in culture for producing endogenous or recombinantcarbohydrate, lipid, nucleic acid, enzymes or protein products or smallmolecules that may be used for subsequent scientific research or asindustrial products; and (iii) use with modern plant tissue culturetechniques to produce transgenic plants or plant tissue cultures thatmay then be used for agricultural research or production. The productionand use of microorganisms such as transgenic plant cells utilizes modernmicrobiological techniques and human intervention to produce a man-made,unique microorganism. In this process, a recombinant DNA is insertedinto a plant cell's genome to create a transgenic plant cell that isseparate and unique from naturally occurring plant cells. Thistransgenic plant cell can then be cultured much like bacteria and yeastcells using modern microbiology techniques and may exist in anundifferentiated, unicellular state. The new plant cell's geneticcomposition and phenotype is a technical effect created by theintegration of a heterologous DNA into the genome of the cell. Anotheraspect of the present disclosure is a method of using a microorganism ofthe present disclosure. Methods of using microorganisms of the presentdisclosure, such as transgenic plant cells, include (i) methods ofproducing transgenic cells by integrating a recombinant DNA into genomeof the cell and then using this cell to derive additional cellspossessing the same heterologous DNA; (ii) methods of culturing cellsthat contain recombinant DNA using modern microbiology techniques; (iii)methods of producing and purifying endogenous or recombinantcarbohydrate, lipid, nucleic acid, enzymes or protein products fromcultured cells; and (iv) methods of using modern plant tissue culturetechniques with transgenic plant cells to produce transgenic plants ortransgenic plant tissue cultures.

As used herein, “progeny” includes any plant, seed, plant cell, and/orregenerable plant part comprising the event DNA derived from an ancestorplant and/or a polynucleotide having at least one of the sequencesprovided as at least 19 consecutive nucleotides of SEQ ID NO:1, at least18 consecutive nucleotides of SEQ ID NO:2, at least 31 consecutivenucleotides of SEQ ID NO:3, at least 31 consecutive nucleotides of SEQID NO:4, at least 51 consecutive nucleotides of SEQ ID NO:5, or at least51 consecutive nucleotides of SEQ ID NO:6. Plants, progeny, and seedsmay be homozygous or heterozygous for the MON87403 event DNA. Progenymay be grown from seeds produced by a plant comprising event MON87403and/or from seeds produced by a plant fertilized with pollen or ovulefrom a plant comprising event MON87403.

Progeny plants may be self-pollinated (also known as “selfing”) togenerate a true breeding line of plants, i.e., plants homozygous for theMON87403 event DNA. Alternatively, progeny plants may be outcrossed,i.e., bred with another plant, to produce a varietal or a hybrid seed orplant. The other plant may be transgenic or nontransgenic. A varietal orhybrid seed or plant of the present disclosure may thus be derived bycrossing a first parent that lacks the specific and unique DNA of eventMON87403 with a second parent comprising event MON87403, resulting in ahybrid comprising the specific and unique DNA of event MON87403. Eachparent can be a hybrid or an inbred/variety, so long as the cross orbreeding results in a plant or seed of the present disclosure, i.e., aseed having at least one allele comprising the specific and unique DNAof event MON87403 and/or at least 19 consecutive nucleotides of SEQ IDNO:1, at least 19 consecutive nucleotides of SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO:5, or at least 51 consecutive nucleotides of SEQ ID NO:6. Twodifferent transgenic plants may thus be mated to produce hybridoffspring that contain two independently segregating, added, transgenes.For example, a plant comprising event MON87403 with increased yield canbe crossed with another transgenic plant, such as one tolerant toglyphosate, to produce a plant having the characteristics of bothtransgenic parents. Selfing of appropriate progeny can produce plantsthat are homozygous for both added transgenes. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in one of several references, e.g., Fehr, in BreedingMethods for Cultivar Development, Wilcox J. ed., American Society ofAgronomy, Madison Wis. (1987). Sexually crossing one plant with anotherplant, i.e., cross-pollinating, may be accomplished or facilitated byhuman intervention, for example: by human hands collecting the pollen ofone plant and contacting this pollen with the style or stigma of asecond plant; by human hands and/or human actions removing, destroying,or covering the stamen or anthers of a plant (e.g., by manualintervention or by application of a chemical gametocide) so that naturalself-pollination is prevented and cross-pollination would have to takeplace in order for fertilization to occur; by human placement ofpollinating insects in a position for “directed pollination” (e.g., byplacing beehives in orchards or fields or by caging plants withpollinating insects); by human opening or removing of parts of theflower to allow for placement or contact of foreign pollen on the styleor stigma; by selective placement of plants (e.g., intentionallyplanting plants in pollinating proximity); and/or by application ofchemicals to precipitate flowering or to foster receptivity (of thestigma for pollen).

In practicing this method, the step of sexually crossing one plant withitself, i.e., self-pollinating or selfing, may be accomplished orfacilitated by human intervention, for example: by human handscollecting the pollen of the plant and contacting this pollen with thestyle or stigma of the same plant and then optionally preventing furtherfertilization of the plant; by human hands and/or actions removing,destroying, or covering the stamen or anthers of other nearby plants(e.g., by detasseling or by application of a chemical gametocide) sothat natural cross-pollination is prevented and self-pollination wouldhave to take place in order for fertilization to occur; by humanplacement of pollinating insects in a position for “directedpollination” (e.g., by caging a plant alone with pollinating insects);by human manipulation of the flower or its parts to allow forself-pollination; by selective placement of plants (e.g., intentionallyplanting plants beyond pollinating proximity); and/or by application ofchemicals to precipitate flowering or to foster receptivity (of thestigma for pollen).

The present disclosure provides a plant part that is derived from aplant comprising event MON87403. As used herein, a “plant part” refersto any part of a plant that is comprised of material directly from orderived from a plant comprising event MON87403. Plant parts include butare not limited to cells, pollen, ovules, kernels, flowers, root or stemtissues, fibers, and leaves. Plant parts may be viable, nonviable,regenerable, and/or non-regenerable.

The present disclosure provides a commodity product that is derived froma plant comprising event MON87403. As used herein, a “commodity product”refers to any composition or product that is comprised of materialderived from a plant, seed, plant cell, or plant part comprising eventMON87403. Commodity products may be sold to consumers and may be viableor nonviable. Nonviable commodity products include but are not limitedto nonviable seeds and grains; processed seeds, seed parts, and plantparts; dehydrated plant tissue, frozen plant tissue, and processed planttissue; seeds and plant parts processed for animal feed for terrestrialand/or aquatic animals consumption, oil, meal, flour, flakes, bran,fiber, milk, cheese, paper, cream, wine, and any other food for humanconsumption; and biomasses and fuel products. Viable commodity productsinclude but are not limited to seeds and plant cells. A plant comprisingevent MON87403 can thus be used to manufacture any commodity producttypically acquired from a corn plant. Any such commodity product that isderived from the plants comprising event MON87403 may contain at least adetectable amount of the specific and unique DNA corresponding to eventMON87403, and specifically may contain a detectable amount of apolynucleotide having a nucleotide sequence of at least 19 consecutivenucleotides of SEQ ID NO:1, at least 18 consecutive nucleotides of SEQID NO:2, at least 31 consecutive nucleotides of SEQ ID NO:3, at least 31consecutive nucleotides of SEQ ID NO:4, at least 51 consecutivenucleotides of SEQ ID NO: 5, or at least 51 consecutive nucleotides ofSEQ ID NO:6. Any standard method of detection for polynucleotidemolecules may be used, including methods of detection disclosed herein.A commodity product is within the scope of the present disclosure ifthere is any detectable amount of at least 19 consecutive nucleotides ofSEQ ID NO:1, at least 18 consecutive nucleotides of SEQ ID NO:2, atleast 31 consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO:5, or at least 51 consecutive nucleotides of SEQ ID NO:6 in thecommodity product.

The plant, progeny, seed, plant cell, plant part (such as pollen, ovule,kernel flower, root or stem tissue, and leaf), and commodity products ofthe present disclosure are therefore useful for, among other things,growing plants for the purpose of producing seed and/or plant partscomprising event MON87403 for agricultural purposes, producing progenycomprising event MON87403 for plant breeding and research purposes, usewith microbiological techniques for industrial and researchapplications, and sale to consumers.

The present disclosure provides methods for producing plants withincreased grain yield and plants comprising event MON87403. EventMON87403-containing plants were produced by an Agrobacterium-mediatedtransformation method, using an inbred corn line with the constructpMON97046 (Table 1 and FIG. 3). Construct pMON97046 contains a plantexpression cassette for expression of the ATHB17 protein in corn plantcells. Transgenic corn cells were regenerated into intact corn plantsand individual plants were selected from the population of independentlytransformed transgenic plants that showed desirable molecularcharacteristics, such as single copy of the transgene cassette at asingle locus, integrity of the transgene cassette, absence of theconstruct backbone sequence, and loss of the unlinked glyphosateresistance selection cassette. Furthermore, inverse PCR and DNA sequenceanalyses were performed to determine the 5′ and 3′ insert-to-plantgenome junctions, to confirm the organization of the elements within theinsert (FIG. 1), and to determine the complete DNA sequence of theinsert in corn event MON87403 (SEQ ID NO:9). In addition, transgenicplants were screened and selected for increased yield under fieldconditions. A corn plant that contains in its genome the MON87403 eventDNA is an aspect of the present disclosure.

Increased yield of a transgenic plant of the present disclosure can bemeasured in a number of ways, including test weight, seed number perplant, seed size, seed weight, seed number per unit area (i.e. seeds, orweight of seeds, per acre), bushels per acre, tons per acre, or kilo perhectare. Expression of the ATHB17 gene in plants comprising eventMON87403 leads to an increase in grain yield. Expression of the ATHB17gene in plants comprising event MON87403 also leads to increased earsize at R1.

Methods for producing a plant with increased yield comprising transgenicevent MON87403 are provided. Transgenic plants used in these methods maybe homozygous or heterozygous for the transgene. Progeny plants producedby these methods may be varietal or hybrid plants; may be grown fromseeds produced by a plant and/or from seed comprising event MON87403produced by a plant fertilized with pollen or ovules from a plantcomprising event MON87403; and may be homozygous or heterozygous for theevent MON87403 DNA. Progeny plants may be subsequently self-pollinatedto generate a true breeding line of plants, i.e., plants homozygous forthe event MON87403 DNA, or alternatively may be outcrossed, i.e., bredwith another unrelated plant, to produce a varietal or a hybrid seed orplant. As used herein, the term “zygosity” refers to the similarity ofDNA at a specific chromosomal location (locus) in a plant. In thepresent disclosure, the DNA specifically refers to the transgene insertalong with the junction sequence (event DNA). A plant is homozygous ifthe transgene insert with the junction sequence is present at the samelocation on each chromosome of a chromosome pair (2 alleles). A plant isconsidered heterozygous if the transgene insert with the junctionsequence is present on only one chromosome of a chromosome pair (1allele). A wild-type plant is null for the event DNA.

A plant with increased yield may be produced by sexually crossing aplant comprising event MON87403 comprising a polynucleotide having thenucleotide sequence of at least 19 consecutive nucleotides of SEQ IDNO:1, at least 18 consecutive nucleotides of SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO:5, or at least 51 consecutive nucleotides of SEQ ID NO:6 withanother plant and thereby producing seed, which is then grown intoprogeny plants. These progeny plants may be analyzed using diagnosticmethods to select for progeny plants that comprise event MON87403 DNA orfor progeny plants with increased yield. The other plant used may or maynot be transgenic. The progeny plant and/or seed produced may bevarietal or hybrid seed.

A plant with increased yield may be produced by selfing a plantcomprising event MON87403 comprising a polynucleotide having thenucleotide sequence of at least 19 consecutive nucleotides of SEQ IDNO:1, at least 18 consecutive nucleotides of SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO: 5, or at least 51 consecutive nucleotides of SEQ ID NO:6 andthereby producing seed, which is then grown into progeny plants. Theseprogeny plants may then be analyzed using diagnostic methods to selectfor progeny plants that comprise event MON87403 DNA.

Progeny plants and seeds encompassed by these methods and produced byusing these methods are distinct from other plants, for example, becausethe progeny plants and seeds comprise a recombinant DNA and as such arecreated by human intervention; contain at least one allele at a specificchromosomal location that comprises the transgenic DNA of the presentdisclosure; and/or contain a detectable amount of a polynucleotidesequence selected from the group consisting of at least 19 consecutivenucleotides of SEQ ID NO:1, at least 18 consecutive nucleotides of SEQID NO:2, at least 31 consecutive nucleotides of SEQ ID NO:3, at least 31consecutive nucleotides of SEQ ID NO:4, at least 51 consecutivenucleotides of SEQ ID NO:5, or at least 51 consecutive nucleotides ofSEQ ID NO:6. A seed may be selected from an individual progeny plant,and so long as the seed comprises SEQ ID NO:1, SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO:5, or at least 51 consecutive nucleotides of SEQ ID NO:6, it willbe within the scope of the present disclosure.

The plants, progeny, seeds, plant cells, plant parts (such as pollen,ovule, kernel flower, root or stem tissue, and leaves), and commodityproducts of the present disclosure may be evaluated for DNA composition,gene expression, and/or protein expression. Such evaluation may be doneby using various methods such as PCR, sequencing, Northern analysis,Southern analysis, Western analysis, immuno-precipitation, and ELISA orby using the methods of detection and/or the detection kits providedherein.

Methods of detecting the presence of compositions specific to eventMON87403 in a sample are provided. One method consists of detecting thepresence of DNA specific to and derived from a cell, a tissue, a seed, aplant or plant parts comprising event MON87403. The method provides fora template DNA sample to be contacted with a primer pair that is capableof producing an amplicon from event MON87403 DNA upon being subjected toconditions appropriate for amplification, particularly an amplicon thatcomprises SEQ ID NO:1, SEQ ID NO:2, at least 31 consecutive nucleotidesof SEQ ID NO:3, at least 31 consecutive nucleotides of SEQ ID NO:4, atleast 51 consecutive nucleotides of SEQ ID NO:5, or at least 51consecutive nucleotides of SEQ ID NO:6, or the complements thereof. Theamplicon is produced from a template DNA molecule derived from eventMON87403, so long as the template DNA molecule incorporates the specificand unique nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO: 5, or at least 51 consecutive nucleotides of SEQ ID NO:6. Theamplicon may be single- or double-stranded DNA or RNA, depending on thepolymerase selected for use in the production of the amplicon. Themethod provides for detecting the amplicon molecule produced in any suchamplification reaction, and confirming within the sequence of theamplicon the presence of the nucleotides corresponding to SEQ ID NO:1,SEQ ID NO:2, at least 31 consecutive nucleotides of SEQ ID NO:3, atleast 31 consecutive nucleotides of SEQ ID NO:4, at least 51 consecutivenucleotides of SEQ ID NO:5, or at least 51 consecutive nucleotides ofSEQ ID NO:6, or the complements thereof. The detection of thenucleotides corresponding to SEQ ID NO:1, SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO:5, or at least 51 consecutive nucleotides of SEQ ID NO:6, or thecomplements thereof in the amplicon are determinative and/or diagnosticfor the presence of event MON87403 specific DNA and thus biologicalmaterial or commodity products comprising event MON87403 in the sample.

Another method is provided for detecting the presence of a DNA moleculecorresponding to SEQ ID NO:7 or SEQ ID NO:8 in a sample consisting ofmaterial derived from plant or plant parts. The method consists of (i)obtaining a DNA sample from a plant or plant part, or from a group ofdifferent plants, (ii) contacting the DNA sample with a DNA probemolecule comprising the nucleotides as set forth in either SEQ ID NO:1or SEQ ID NO:2, (iii) allowing the probe and the DNA sample to hybridizeunder stringent hybridization conditions, and then (iv) detecting ahybridization event between the probe and the target DNA sample.Detection of the hybrid composition is diagnostic for the presence ofSEQ ID NO:7 or SEQ ID NO:8, as the case may be, in the DNA sample.Absence of hybridization is alternatively diagnostic of the absence ofthe transgenic event in the sample if the appropriate positive controlsare run concurrently. Alternatively, determining that a particular plantor plant part comprises either or both of the sequences corresponding toSEQ ID NO:1 or SEQ ID NO:2, or the complements thereof, is determinativethat the plant or plant part comprises at least one allele correspondingto event MON87403.

It is thus possible to detect the presence of a nucleic acid molecule ofthe present disclosure by any well known nucleic acid amplification anddetection methods such as polymerase chain reaction (PCR) or another DNAamplification method, or DNA hybridization using nucleic acid probes. Anevent-specific PCR assay is discussed, for example, by Taverniers et al.(J. Agric. Food Chem., 53: 3041-3052, 2005) in which an event-specifictracing system for transgenic maize lines Bt11, Bt176, and GA21 and fortransgenic event RT73 is demonstrated. In this study, event-specificprimers and probes were designed based upon the sequences of thegenome/transgene junctions for each event. Transgenic plant eventspecific DNA detection methods have also been described in U.S. Pat.Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974; 6,689,880; 6,900,014;and 6,818,807.

DNA detection kits are provided. One type of kit contains at least oneDNA molecule of sufficient length of contiguous nucleotides of SEQ IDNO:7, SEQ ID NO:9, or SEQ ID NO:10 to function as a DNA primer or probespecific for detecting the presence of DNA derived from transgenic eventMON87403 in a sample. The DNA molecule being detected with the kitcomprises contiguous nucleotides of the sequence as set forth in SEQ IDNO:1, at least 31 consecutive nucleotides of SEQ ID NO:3, or at least 51consecutive nucleotides of SEQ ID NO:5. Alternatively, the kit maycontain at least one DNA molecule of sufficient length of contiguousnucleotides of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 to function asa DNA primer or probe specific for detecting the presence of DNA derivedfrom transgenic event MON87403 in a sample. The DNA molecule beingdetected with the kit comprises contiguous nucleotides as set forth inSEQ ID NO:2, at least 31 consecutive nucleotides of SEQ ID NO:4, or atleast 51 consecutive nucleotides of SEQ ID NO:6. The kit of theinvention may optionally include instructions, means for increasingconvenience of using the kit such as buffers and test tubes, and thelike.

An alternative kit employs a method in which the target DNA sample iscontacted with a primer pair as described above, then performing anucleic acid amplification reaction sufficient to produce an ampliconcomprising at least 19 consecutive nucleotides of SEQ ID NO:1, at least18 consecutive nucleotides of SEQ ID NO:2, at least 31 consecutivenucleotides of SEQ ID NO:3, at least 31 consecutive nucleotides of SEQID NO:4, at least 51 consecutive nucleotides of SEQ ID NO: 5, or atleast 51 consecutive nucleotides of SEQ ID NO:6. Detection of theamplicon and determining the presence of the consecutive nucleotides ofSEQ ID NO:1, the consecutive nucleotides of SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO: 5, or at least 51 consecutive nucleotides of SEQ ID NO:6, or thecomplements thereof within the sequence of the amplicon is diagnosticfor the presence of event MON87403 specific DNA in a DNA sample.

A DNA molecule sufficient for use as a DNA probe is provided that isuseful for determining, detecting, or for diagnosing the presence oreven the absence of DNA specific and unique to event MON87403 DNA in asample. The DNA molecule contains the consecutive nucleotides of SEQ IDNO:1, or the complement thereof, the consecutive nucleotides of SEQ IDNO:2, or the complement thereof, at least 31 consecutive nucleotides ofSEQ ID NO:3, or the complement thereof, at least 31 consecutivenucleotides of SEQ ID NO:4, or the complement thereof, at least 51consecutive nucleotides of SEQ ID NO:5, or the complement thereof, or atleast 51 consecutive nucleotides of SEQ ID NO:6, or the complementthereof.

Nucleic acid amplification can be accomplished by any of the variousnucleic acid amplification methods known in the art, including thermaland isothermal amplification methods. The sequence of the heterologousDNA insert, junction sequences, or flanking sequences from eventMON87403 can be verified by amplifying such sequences from the eventusing primers derived from the sequences provided herein followed bystandard DNA sequencing of the amplicon or of the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microwell plate. Following thermal amplification of theregion of interest (using one primer to the inserted sequence and one tothe adjacent flanking genomic sequence), a single-stranded amplicon canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. Detection of a fluorescent or other signal indicates thepresence of the insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Another method is the pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to a single-strandedamplicon from the region of interest (one primer to the insertedsequence and one to the flanking genomic sequence) and incubated in thepresence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. ddNTPs are added individuallyand the incorporation results in a light signal which is measured. Alight signal indicates the presence of the transgene insert/flankingsequence due to successful amplification, hybridization, and single ormulti-base extension.

Fluorescence polarization as described by Chen, et al. (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon.Using this method an oligonucleotide is designed which overlaps thegenomic flanking DNA and the inserted DNA junction. The oligonucleotideis hybridized to single-stranded amplicon from the region of interest(one primer to the inserted DNA and one to the flanking genomic DNAsequence) and incubated in the presence of a DNA polymerase and afluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

TAQMAN® (PE Applied Biosystems, Foster City, Calif.) may also be used todetect and/or to quantify the presence of a DNA sequence using theinstructions provided by the manufacturer. Briefly, a FREToligonucleotide probe is designed which overlaps the genomic flankingDNA and the insert DNA junction. The FRET probe and amplificationprimers (one primer to the insert DNA sequence and one to the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful amplificationand hybridization.

Molecular beacons have been described for use in sequence detection asdescribed in Tyangi et al. (Nature Biotech. 14:303-308, 1996). Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe andamplification primers (one primer to the insert DNA sequence and one tothe flanking genomic sequence) are cycled in the presence of athermostable polymerase and dNTPs. Following successful amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties, which leads to the production of afluorescent signal. The fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Other described methods, such as microfluidics (U.S. Patent PublicationNo. 2006/068398; U.S. Pat. No. 6,544,734) provide methods and devices toseparate and amplify DNA samples. Optical dyes are used to detect andmeasure specific DNA molecules (WO/05017181). Nanotube devices(WO/06024023) that comprise an electronic sensor for the detection ofDNA molecules or nanobeads that bind specific DNA molecules can then bedetected.

DNA detection kits can be developed using the compositions disclosedherein and the methods well-known in the art of DNA detection. The kitsare useful for the identification of event MON87403 in a sample and canbe applied to methods for breeding plants containing the appropriateevent DNA. The kits may contain DNA primers or probes that are similaror complementary to SEQ ID NO:1-6, or fragments or complements thereof.

The kits and detection methods of the present disclosure are thereforeuseful for, among other things, identifying event MON87403, selectingplant varieties or hybrids comprising event MON87403, detecting thepresence of DNA derived from event MON87403 in a sample, and monitoringsamples for the presence and/or absence of event MON87403 or plants,plant parts or commodity products comprising event MON87403.

The following examples are included to demonstrate examples of certainembodiments of the disclosure. It should be appreciated by those ofskill in the art that the techniques disclosed in the examples thatfollow represent approaches the inventors have found function well inthe practice of the disclosure, and thus can be considered to constituteexamples of preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments that are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the disclosure.

EXAMPLES Example 1: Transformation of Corn with pMON97046 and EventSelection

This example describes transformation and generation of transgenic cornevents, and selection of event MON87403.

An Agrobacterium-mediated transformation method was used to transformcorn cells with a plasmid vector designated as pMON97046. The constructcomprised a right border, an ATHB17 expression cassette, a left border,and a CP4 expression cassette as shown in FIG. 3, as well as othergenetic elements necessary for replication and selection in bacteria.The ATHB17 cassette in the transgenic insert as set forth in SEQ ID NO:9contained the HB17 coding region from Arabidopsis thaliana regulated bya CaMV 35S enhancer, a rice actin 1 promoter, a wheat chlorophyll a/bbinding protein leader, a rice actin 1 intron, and a hsp17 3′polyadenylation sequence. ATHB17 belongs to the homeodomain leucinezipper (HD-Zip) class II gene family. The CP4 cassette contained a CP4coding region regulated by a rice actin promoter, leader and intron, anda NOS 3′ polyadenylation sequence. The CP4 cassette was used as aselectable marker. Table 1 is a summary of the genetic elements inpMON97046.

TABLE 1 Summary of the genetic elements in pMON97046 Location Genetic inPlasmid Element Vector Function (Reference) T-DNA B¹-Right Border  1-357DNA region from Agrobacterium tumefaciens Region containing the rightborder sequence used for transfer of the T-DNA (Depicker et al., 1982;Zambryski et al., 1982) Intervening Sequence 358-375 Sequence used inDNA cloning P²-e35S/Ract1  376-1556 Chimeric promoter consisting of theduplicated enhancer region from the cauliflower mosaic virus 35S RNApromoter (CaMV) (Kay et al., 1987) combined with the promoter of theact1 gene from Oryza sativa that encodes Actin 1 (McElroy et al., 1990)that directs transcription in plant cells Intervening Sequence 1557-1561Sequence used in DNA cloning L³-Cab 1562-1622 5′ UTR leader sequencefrom chlorophyll a/b- binding (CAB) protein of Triticum aestivum (wheat)that is involved in regulating gene expression Intervening Sequence1623-1638 Sequence used in DNA cloning I⁴-Ract1 1639-2118 Intron andflanking UTR sequence of the act1 gene from Oryza sativa (rice) encodingrice Actin 1 protein (McElroy et al., 1991). This sequence is involvedin regulating gene expression (McElroy et al., 1991) InterveningSequence 2119-2130 Sequence used in DNA cloning CS⁵-ATHB17 2131-2958Coding sequence of the ATHB17 gene from Arabidopsis thaliana encoding amember of the class II homeodomain-leucine zipper gene family (HD-ZipII) that is thought to act as a transcription factor (Ariel et al.,2007) Intervening Sequence 2959-2971 Sequence used in DNA cloningT⁶-Hsp17 2972-3181 3′ UTR sequence from a heat shock protein, Hsp17, ofTriticum aestivum (wheat) (McElwain and Spiker, 1989) that directspolyadenylation of the mRNA Intervening Sequence 3182-3234 Sequence usedin DNA cloning B-Left Border 3235-3676 DNA region from Agrobacteriumtumefaciens Region containing the left border sequence used for transferof the T-DNA (Barker et al., 1983) Backbone Intervening Sequence3677-3682 Sequence used in DNA cloning P-Ract1 3683-4603 Promoter andleader of the act1 gene from Oryza sativa (rice) encoding the rice Actin1 protein (McElroy et al., 1990) that directs transcription in plantcells I-Ract1 4604-5081 Intron and flanking UTR sequence of the act1gene from Oryza sativa (rice) encoding rice Actin 1 protein (McElroy etal., 1991). This sequence is involved in regulateing gene expressionIntervening Sequence 5082-5090 Sequence used in DNA cloning TS⁷-CTP25091-5318 Targeting sequence of the ShkG gene from Arabidopsis thalianaencoding the EPSPS transit peptide region that directs transport of theprotein to the chloroplast (Herrmann, 1995; Klee et al., 1987) CS-cp4epsps 5319-6686 Coding sequence of the aroA gene from Agrobacterium sp.strain CP4 encoding the CP4 EPSPS protein that provides herbicidetolerance (Barry et al., 2001); (Padgette et al., 1996) InterveningSequence 6687-6701 Sequence used in DNA cloning T-nos 6702-6954 3′ UTRsequence of the nopaline synthase (nos) gene from Agrobacteriumtumefaciens pTi encoding NOS (Bevan et al., 1983; Fraley et al., 1983),that directs polyadenylation of the mRNA Intervening Sequence 6955-7005Sequence used in DNA cloning OR⁸-ori V 7006-7402 Origin of replicationfrom the broad host range plasmid RK2, used for maintenance of plasmidin Agrobacterium (Stalker et al., 1981) Intervening Sequence 7403-8910Sequence used in DNA cloning CS-rop 8911-9102 Coding sequence forrepresser of primer protein from the ColE1 plasmid for maintenance ofplasmid copy number in E. coli (Giza and Huang, 1989) InterveningSequence 9103-9529 Sequence used in DNA cloning OR-ori-pUC  9530-10118Origin of replication from plasmid pUC for maintenance of plasmid in E.coli (Vieira and Messing, 1987) Intervening Sequence 10119-10648Sequence used in DNA cloning aadA 10649-11537 Bacterial promoter, codingsequence, and 3′ UTR for an aminoglycoside-modifying enzyme,3″(9)-O-nucleotidyltransferase from the transposon Tn7 (Fling et al.,1985). This sequence confers spectinomycin and streptomycin resistanceIntervening Sequence 11538-11673 Sequence used in DNA cloning ¹B, Border²P, Promoter ³L, Leader ⁴I, Intron ⁵CS, Coding Sequence ⁶T,Transcription Termination Sequence ⁷TS, Targeting Sequence ⁸OR, Originof Replication

After transformation with construct pMON97046, transformed cells wereallowed to grow and multiply on selective media. Plants were regeneratedfrom surviving cells. A total of 2687 independent R0 transformationevents were produced and characterized for insert copy number andlinkage of ATHB17 with the CP4 cassette. The R1 plant tissue was usedfor further molecular characterization on insert copy number, linkage tothe CP4 expression cassette and the presence of the transformationvector backbone. Events at R3 generation were screened for copy number(the number of copies of the T-DNA within one locus), the integrity ofthe inserted cassette, the absence of backbone sequence, genomiclocation of the insert, and expression of the ATHB17 transgenetranscript. Events with undesirable phenotypes or molecularcharacteristics, such as presence of multiple copies of the transgeneand/or molecular complexity of the insert, the presence of thetransformation vector backbone sequence, and insertion of the transgenein an intragenic region, were eliminated. Furthermore, linkage Southernanalysis was done to remove events where the ATHB17 expression cassettewas linked to the CP4 selectable marker cassette. No additional elementsfrom the original transformation vector, linked or unlinked to theintact cassettes, were identified in the genome of these events. A totalof 324 events met the basic molecular selection criteria based on the R0analyses and were advanced to R1 generation. Subsequently, 74 eventswere advanced to the R2 generation, 67 events were advanced to year-1field trials, 22 events were advanced to the R3 generation, and 15events were advanced to year-2 field testing. FIG. 2 show comparativedata for 14 individual events advanced to field testing in 2009. It wasadditionally shown that a 155-nt deletion of the genomic DNA occurredupon integration of the MON87403 T-DNA.

Example 2: Isolation of Flanking Sequences Using Inverse PCR andIdentification of Flanking Sequences by Sequencing

This example describes isolation of the corn genomic DNA sequencesflanking the transgenic DNA insert using inverse PCR for event MON87403,and identification of the flanking genomic sequences by sequencing.

Sequences flanking the T-DNA insertion in event MON87403 were determinedusing inverse PCR. The PCR procedure was followed using genomic DNA fromplants comprising event MON87403, using methods known in the art.Primers were located within the Agrobacterium-left border sequence andwere SQ6164 (SEQ ID NO:19) and SQ13205 (SEQ ID NO:20), used in thatorder along with SQ22459 (SEQ ID NO:23) and SQ22458 (SEQ ID NO:22).Amplicons were detected on a 1% TAE agarose gel and samples weresequenced using left border primer SQ6165 (SEQ ID NO:21). The sameprocedure was followed for the isolation of the right border flank,using primers SQ21173 (SEQ ID NO:24) and SQ22464 (SEQ ID NO:25) forprimary, primers SQ22460 (SEQ ID NO:26) and SQ22465 (SEQ ID NO:27) forsecondary, and primers SQ22461 (SEQ ID NO:28) and SQ22471 (SEQ ID NO:29)for the tertiary round of PCR.

Amplicons obtained by inverse PCR procedure for the Left and the RightBorder reaction for corn event MON87403 were sequenced. The subsequentalignment of the amplicon sequences produced approximately 531 bp offlanking sequence 5′ to the Right Border and 277 bp 3′ to the leftborder sequence of the T-DNA.

Short flank sequences (RB and LB flanks) obtained by inverse PCR weresubjected to BLAST analysis. The matched genomic sequences wereelectronically extended along the corn genome beyond 1 kb to obtainvirtual extended flanks. The virtually extended flank sequence was thenused to design primers for genomic PCR and confirm the actual genomicsequence.

The flanking sequence and wild type sequences were used to designprimers for TAQMAN® endpoint assays used to identify the events anddetermine zygosity as described in Example 3.

Example 3: Event-Specific Endpoint TAQMAN® and Zygosity Assays

This example describes an event-specific endpoint TAQMAN® thermalamplification method for identification of event MON87403 DNA in asample.

Examples of conditions useful with the event MON87403-specific endpointTAQMAN® method are described in Tables 2 and 3. The DNA primers used inthe endpoint assay are primers SQ23846 (SEQ ID NO:11) and SQ4603 (SEQ IDNO:12) and the 6-FAM™ labeled oligonucleotide probe is PB10644 (SEQ IDNO:13). 6FAM™ is a fluorescent dye product of Applied Biosystems (FosterCity, Calif.) attached to the DNA probe. For TAQMAN® MGB (Minor GrooveBinding) probes, the 5′ exonuclease activity of Taq DNA polymerasecleaves the probe from the 5′-end, between the fluorophore and quencher.When hybridized to the target DNA strand, quencher and fluorophore areseparated enough to produce a fluorescent signal.

Primers SQ23846 (SEQ ID NO:11) and SQ4603 (SEQ ID NO:12), when used asdescribed with probe PB10644 (SEQ ID NO:13), produce an amplicon that isdiagnostic for event MON87403 DNA. The analysis includes a positivecontrol from corn known to comprise event MON87403 DNA, a negativecontrol from a wild type corn or a corn not comprising event 87403 DNA,and a negative control that contains no template DNA.

These assays are optimized for use with Applied Biosystems GeneAmp PCRSystem 9700, ABI 9800 Fast Thermal Cycler and MJ Opticon. Other methodsand apparatus known to those skilled in the art may be used to produceamplicons that identify the event MON87403 DNA.

TABLE 2 Corn MON87403 Event-Specific Zygosity Endpoint TAQMAN ® PCRConditions Step Reagent Volume Comments 1 18 megohm water adjust forfinal volume of 10 μl 2 2X Universal Master Mix 5.0 μl 1X final (dNTPs,enzyme and buffer) concentration of dNTPs, enzyme and buffer 3 EventPrimers SQ23846 and 0.5 μl 1.0 μM final SQ4603 Mix (resuspended inconcentration 18 megohm water to a concentration of 20 μM for eachprimer) Example: In a microcentrifuge tube, the following are added toachieve: 500 μl at a final concentration of 20 μM 100 μl of PrimerSQ23846 at a concentration of 100 μM 100 μl of Primer SQ4603 at aconcentration of 100 μM 300 μl of 18 megohm water 4 Event 6-FAM ™ MGBProbe 0.2 μl 0.2 μM final PB10644 (resuspended in 18 concentrationmegohm water to a concentration of 10 μM) 5 Wild Type Primer SQ25061 0.5μl 1 μM final and Wild Type Primer concentration SQ25062 Mix(resuspended in 18 megohm water to a concentration of 20 μM for eachprimer) 6 Wild Type VIC ™ Probe 0.2 μl 0.2 μM final PB10866 (resuspendedin 18 concentration megohm water to a concentration of 10 μM) 7Extracted DNA (template): 3.0 μl 1. Leaf or seed samples to be analyzed2. Negative control (non-event MON87403 DNA) 3. Negative water control(no template control) 4. Positive control (MON87403 DNA)

TABLE 3 Endpoint Zygosity TAQMAN ® thermocycler conditions Cycle No.Settings 1 50° C. 2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds 64°C. 1 minute (−1° C./cycle) 30 95° C. 15 seconds 54° C. 1 minute 1 10° C.Forever

The following example describes an event-specific endpoint TAQMAN®thermal amplification method developed to determine the zygosity ofevent MON87403 in a sample. A zygosity assay is useful for determiningif a plant comprising an event is homozygous, heterozygous or null forthe event DNA. An event-comprising plant is homozygous for the event DNAif the transgenic DNA is present at the same location on each chromosomeof a chromosomal pair. An event-comprising plant is heterozygous for theevent DNA if the transgenic DNA is present on only one chromosome of achromosomal pair. A plant is null for the event DNA if the plant doesnot contain the event DNA; that is, the plant is wild type for thelocus. A set of primers (SEQ ID NOs:11 and 12, and SEQ ID NOs:14 and15), a 6FAM™ labeled probe (SEQ ID NO:13), and a VIC™ labeled probe (SEQID NO: 16) were used in the assays. These primers are diagnostic. Inthis example, primers SEQ ID NOs:11 and 12 and the 6FAM™ labeledoligonucleotide probe SEQ ID NO:13 produce a DNA amplicon revealed bythe liberation of a fluorescent signal from probe SEQ ID NO:13, which isdiagnostic for event MON87403 DNA, indicating at least a copy of theinserted transgenic DNA present in the genomic DNA. Primers SEQ IDNOs:14 and 15 and the VIC™ labeled oligonucleotide probe SEQ ID NO:16produce an amplicon revealed by the liberation of a fluorescent signalfrom probe SEQ ID NO:16, which is diagnostic for the wild type allele,indicating no copy of the inserted transgenic DNA present in the genomicDNA. When the primers and probes are mixed together in a PCR with DNAextracted from a plant, release of a fluorescent signal only from the6FAM™ labeled oligonucleotide probe (SEQ ID NO:13) is indicative of anddiagnostic of a plant homozygous for event MON87403. When the primersand probes are mixed together in a PCR with DNA extracted from a plant,release of a fluorescent signal from both the 6FAM™ labeledoligonucleotide probe SEQ ID NO:13 and the VIC™ labeled oligonucleotideprobe SEQ ID NO:16 is indicative of and diagnostic of a plantheterozygous for event MON87403. When the primers and probes are mixedtogether in a PCR with DNA extracted from a plant, release of afluorescent signal from only the VIC™ labeled oligonucleotide probe SEQID NO:16 is indicative of and diagnostic of a plant null for eventMON87403, i.e. wild type. The assays also include a positive controlfrom corn containing event MON87403 DNA, a negative control from a wildtype corn or corn not comprising event 87403 DNA and a negative controlthat contains no template DNA.

TABLE 4 Examples of Primer And Probe Combinations Usedfor Endpoint and Zygosity Assays SEQ Type Direction ID NO SequencesPrimers Reverse 11 TGCTCTGTATCCTCCAC CATGT Forward 12 TTTCTCCATATTGACCATCATACTCAT Probe MON87403 13 6FAM- allele CTGATCCACATTTCC PrimersReverse 14 GCATGTCTTTAAAAATC CTTGGTTTAC Forward 15 TGATGTTTTTACTGGATTGCATTACC Probe Wild type 16 VIC- allele CACCCTAAGAGTACTA TTGAAGA

Example 4: Detection of Event MON87403

This example describes how one may identify the MON87403 event withinprogeny of any breeding event containing MON87403 corn.

Event DNA primer pairs are used to produce an amplicon diagnostic forcorn event MON87403. An amplicon diagnostic for MON87403 comprises atleast one junction sequence, provided herein as SEQ ID NO:1, SEQ IDNO:2, at least 31 consecutive nucleotides of SEQ ID NO:3, at least 31consecutive nucleotides of SEQ ID NO:4, at least 51 consecutivenucleotides of SEQ ID NO:5, or at least 51 consecutive nucleotides ofSEQ ID NO:6, or the complements thereof ([A] or [B], respectively asillustrated in FIG. 1).

Event primer pairs that produce an amplicon diagnostic for MON87403include primer pairs designed using the flanking sequences and theintegrated transgenic DNA sequence. To acquire a diagnostic amplicon inwhich at least 19 nucleotides of SEQ ID NO:1 is found, one would designa forward primer based upon the 5′ flanking sequence of SEQ ID NO:10from bases 1 through 1335 and a reverse primer based upon the insertedexpression cassette DNA sequence, SEQ ID NO:9, or from positions 1356through 4477 of SEQ ID NO:10, in which the primers are of sufficientlength of contiguous nucleotides to specifically hybridize to SEQ IDNO:10 on either side of the junction sequence. To acquire a diagnosticamplicon in which at least 18 nucleotides of SEQ ID NO:2 is found, onewould design a forward primer based upon the inserted expressioncassette DNA sequence, SEQ ID NO:9, or from positions 1346 through 4467of SEQ ID NO:10 and a reverse primer based upon the 3′ flanking sequenceof SEQ ID NO:10, from bases 4488 through 5744, in which the primers areof sufficient length of contiguous nucleotides to specifically hybridizeto SEQ ID NO:10 on either side of the junction sequence. For practicalpurposes, one should design primers that produce amplicons of a limitedsize range, for example, between 100 to 1000 bases. Smaller sized(shorter nucleotide length) amplicons in general may be more reliablyproduced in PCR reactions, allow for shorter cycle times and be easilyseparated and visualized on agarose gels or adapted for use in endpointTAQMAN®-like assays. In addition, amplicons produced using primer pairscan be cloned into vectors, propagated, isolated and sequenced or can besequenced directly with methods well-established in the art. Any primerpair derived from the combination of flanking sequences and theintegrated transgenic DNA sequence from SEQ ID NO:10 that are useful ina DNA amplification method to produce an amplicon diagnostic forMON87403, plants comprising MON87403 or progeny thereof is an aspect ofthe present disclosure. Any single isolated DNA primer moleculecomprising a sufficient length of contiguous nucleotides, for instance,at least 11 contiguous nucleotides of SEQ ID NO:10, or its complementthat is useful in a DNA amplification method to produce an amplicondiagnostic for MON87403, plants comprising MON87403 or progeny thereofis an aspect of the present disclosure. In another embodiment, thepresent disclosure provides isolated DNA primer molecules diagnostic forMON87403 comprising a combination of flanking sequences and integratedtransgenic DNA sequence from SEQ ID NO:7 or 8.

An example of the amplification conditions for this analysis isillustrated in Tables 2 and 3. However, any modification of thesemethods or the use of DNA primers homologous or complementary to SEQ IDNO:7, 8, and 10 that produce an amplicon diagnostic for MON87403 iswithin the scope of the present disclosure. A diagnostic ampliconcomprises a DNA molecule homologous or complementary to at least onetransgene/genomic junction DNA (SEQ ID NO:1, SEQ ID NO:2, at least 31consecutive nucleotides of SEQ ID NO:3, at least 31 consecutivenucleotides of SEQ ID NO:4, at least 51 consecutive nucleotides of SEQID NO:5, or at least 51 consecutive nucleotides of SEQ ID NO:6, or thecomplements thereof), or a substantial portion thereof.

An analysis for event MON87403 DNA in a sample should include a positivecontrol from event MON87403, a negative control from a corn plant thatdoes not contain event MON87403, for example, but not limited to wildtype control, and a negative control that contains no corn genomic DNA.A primer pair that will amplify an endogenous corn DNA molecule willserve as an internal control for the DNA amplification conditions.Additional primer sequences can be selected from SEQ ID NOs:7, 8, and 10by those skilled in the art of DNA amplification methods, and conditionsselected for the production of an amplicon by the methods shown inTables 2 and 3 may differ, but result in an amplicon diagnostic forevent MON87403 DNA. The use of these DNA primer sequences withmodifications to the methods of Table 2 and 3 are within the scope ofthe disclosure. The amplicon produced by at least one DNA primersequence derived from SEQ ID NOs:7, 8, and 10 that is diagnostic forMON87403 is an aspect of the disclosure.

DNA detection kits that contain at least one DNA primer derived from SEQID NOs:7, 8, and 10 that when used in a DNA amplification method,produces a diagnostic amplicon for MON87403, plants comprising MON87403or progeny thereof is an aspect of the disclosure. A corn plant or seed,wherein its genomic DNA produces an amplicon diagnostic for MON87403when tested in a DNA amplification method is an aspect of thedisclosure. The assay for the MON87403 amplicon can be performed byusing an Applied Biosytems GeneAmp PCR System 9700, ABI 9800 FastThermal Cycler and MJ Opticon, Stratagene Robocycler, MJ Engine,Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient thermocycler or anyother amplification system that can be used to produce an amplicondiagnostic of MON87403.

A representative sample of seed comprising event MON87403 disclosedabove and recited in the claims has been deposited according to theBudapest Treaty with the ATCC. The date of deposit was Mar. 1, 2013, andthe ATCC accession number is PTA-13584. Upon issuance of a patent, allrestrictions upon the deposit will be removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §§ 1.801-1.809.The deposit will be maintained in the depository for a period of 30years, or 5 years after the last request, or for the effective life ofthe patent, whichever is longer, and will be replaced as necessaryduring that period.

Example 5: Plants Comprising the MON87403 Event Exhibit Increased Yield

Plants comprising the MON87403 event were planted over multiplelocations in multiple testers for several years under optimal productionmanagement practices and maximum weed and pest control. The data in FIG.4 show that plants comprising the MON87403 event exhibited a mean yieldadvantage across locations in all but one year, as compared to theappropriate control plants.

Example 6: Plants Comprising the MON87403 Event Exhibit Increased R1 EarWeight

To investigate the effect of expression of the ATHB17 gene on the R1 earweight, data were collected from field trials conducted within the majorU.S. maize production region. Plants comprising the MON87403 event werecompared to a near isogenic conventional control across 13 U.S.locations in 2012 for comparison of the ear weight at the R1 stage. Theexperiment was planted in a randomized complete block design with 4replications per site. Data was collected from 13 sites (N=51). Plantswere sampled by separating the primary ear from the plant. Ears weredried in a forced air oven at 80° C. until constant weight prior toweighing. A statistically significant increase in R1 ear weight wasobserved in plants comprising the MON87403 event compared to theconventional control in a combined site analysis (Table 5).

TABLE 5 Combined site analysis of R1 ear weight from plants comprisingthe MON87403 event and the conventional control from U.S. field trialsCharacteristic MON 87403 Control (units) (Mean ± SE) (Mean ± SE)Difference p-value R1 ear weight (g) 144.5 (±8.5) 129.3 (±8.1) 15.20.004

Having illustrated and described the principles of the presentdisclosure, it should be apparent to persons skilled in the art that thedisclosure can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

What is claimed is:
 1. A pair of DNA molecules comprising a first DNAmolecule and a second DNA molecule different from the first DNAmolecule, wherein said first and second DNA molecules each comprise anucleotide sequence of sufficient length of contiguous nucleotides ofSEQ ID NO:10, or a full complement thereof, to function as DNA primerswhen used together in an amplification reaction with DNA from eventMON87403 to produce an amplicon diagnostic for transgenic corn eventMON87403 DNA in a sample, wherein the amplicon comprises a nucleotidesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQID NO:8.
 2. A method of detecting the presence of a DNA molecule from atransgenic corn plant comprising event MON87403 in a sample, said methodcomprising: (a) contacting said sample with the pair of DNA molecules ofclaim 1; (b) performing an amplification reaction sufficient to producea DNA amplicon comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; and (c) detecting thepresence of said DNA amplicon in said reaction, wherein the presence ofsaid DNA amplicon in said reaction indicates the presence of a DNAmolecule from a transgenic corn plant comprising event MON87403 in saidsample.
 3. A DNA detection kit comprising: (a) the pair of DNA moleculesof claim 1; and (b) a DNA probe comprising a nucleotide sequence ofsufficient length of contiguous nucleotides of SEQ ID NO:10, or a fullcomplement thereof, to function as a DNA probe that hybridizes understringent hybridization conditions with a DNA molecule comprising anucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8 and SEQ ID NO:10 and does not hybridize under thestringent hybridization conditions with a DNA molecule not comprising anucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8 and SEQ ID NO:10.
 4. The pair of DNA molecules ofclaim 1, wherein the amplicon comprises the nucleotide sequence of SEQID NO:1.
 5. The pair of DNA molecules of claim 1, wherein the ampliconcomprises the nucleotide sequence of SEQ ID NO:2.
 6. The pair of DNAmolecules of claim 1, wherein the first DNA molecule comprises thenucleotide sequence of SEQ ID NO:11 and wherein the second DNA moleculecomprises the nucleotide sequence of SEQ ID NO:12.